Urethritis is the most common male reproductive tract syndrome, yet once gonorrhea is accounted for, up to 50% of non-gonococcal urethritis (NGU) cases have no known etiology, suggesting the presence of occult pathogens. New metagenomic approaches have been extremely effective in identifying previously unidentified organisms. Broad range ribosomal RNA gene PCR is a powerful technique for bacterial pathogen discovery, but is less sensitive than targeted species-specific PCR; thus both approaches are required. Using these techniques, Fredricks and colleagues recently identified 16 newly recognized bacteria among women with bacterial vaginosis (BV) by broad range rRNA gene PCR. Using more sensitive species-specific quantitative PCR (qPCR) assays, 7 of these novel bacteria were significantly associated with BV (Fredricks, NEJM 2005) and 5 (BV-associated bacterium [BVAB] 2, BVAB3, Leptotrichia/Sneathia, Atopobium vaginae, and Megasphaera type 1) were associated with cervicitis, making them strong candidate urethral pathogens. Given the relatively common nature of NGU, the high proportion of unexplained cases and the possibility of multiple etiologies, we propose a 2-tiered approach to identify novel urethral pathogens within a recently completed case-control study. In pathogen discovery activities we propose to use broad range 16S rRNA gene PCR with pyrosequencing to describe and compare the urethral bacterial biota in 136 men with idiopathic urethritis to that in 136 men without urethritis. In a more directed approach, we propose to describe the epidemiology of 5 novel candidate bacterial pathogens (BVAB2, BVAB3, Leptotrichia/Sneathia species, Megasphaera type 1, A. vaginae) in men using species-specific qPCR assays, focusing on evaluating their association with NGU. We will also evaluate behavioral and clinical characteristics associated with detection of novel bacteria, including the clinical response of any organisms associated with NGU to the standard antimicrobial therapies for NGU.